The intensity of fluorescence can be decreased by a wide variety of processes overall called quenching. For probes with a stem structure, e.g. molecular beacons and scorpion probes, or other pair of probes, e.g. kissing probes and competitive hybridisation probes, where the reporter dye and quencher are brought into close proximity, the main mechanism of quenching is static or contact quenching where the energy from the fluorophore reporter dye is transferred to the quencher which then dissipates the energy as heat without fluorescence (J. R. Lakowicz, Principles of fluorescence spectroscopy, 1999, Kluwer Academic/Plenum Publishers, New York). Static quenching occurs through the formation of a ground state complex where the reporter dye and quencher moieties bind together to form a ground state complex—an intramolecular dimer. Förster resonance energy transfer (FRET or RET) is the mechanism which commonly is cited as controlling fluorescence quenching in probes without a stem structure, e.g. TaqMan probes (Marras et. al., Nucleic Acid Res., 2002, 30, e122). FRET is a process that occurs whenever the emission spectrum of a fluorophore overlaps with the absorption spectrum of the acceptor molecule e.g. the quencher (J. R. Lakowicz, ibid.). For optimal FRET quenching the reporter and quencher moieties should be chosen so the spectral overlap between the emission fluorescence and the quencher absorption is optimal. The efficiency of the process is dependent on 1/r6 (Förster distance) where r is the fluorophore—quencher distance (T. Förster, Ann. Phys., 1948, 2, 55). In another collisional quenching mechanism (Dexter quenching) the excited state fluorophore is deactivated during a diffusive encounter with the quencher. Traditionally, dual-labelled probes have been designed focusing on choosing a matched fluorophore—quencher pair for optimal FRET quenching. It has recently been proposed that fluorophore—quencher pairs in non-stem dual labelled probes also should be chosen for increasing static quenching through the formation of the ground-state complex—the intramolecular dimer—as this was found to be an extremely effective quenching method (Johansson et al, J. Am. Chem. Soc., 2002, 124, 6950-6956; Johansson and Cook, Chem. Eur. J., 2003, 9, 3466-3471). Thus, fluorophore—quencher pairs with an inherent affinity of each other are suited for ground-state complex formation resulting potentially in increased quenching. Quenchers can be fluorescent e.g. TAMRA (tetramethylrhodamine) or non-fluorescent e.g. derivatives of 4-(dimethylamino)azobenzene (Dabcyl). Non-fluorescent quenchers are also referred to as dark quenchers. Fluorescent quenchers have 2 significant limitations: The presence of background fluorescence and the preclusion of detection of reporter fluorescence at the acceptor fluorescence emission. Thus, preferred quenchers are dark quenchers.
1,4-Diaminoanthraquinones have earlier been incorporated into oligodeoxynucleotides using phosphoramidite chemistry. The 1,4-diaminoanthraquinone dark quencher LQ1 (“LQ1”) was incorporated into the 5′-end of DNA molecular beacons (May et. al., Chem. Commun., 2003, 970-971) with either FAM or Cy5 as a fluorophore. The LQ1 quencher has an absorption range of 500-700 nm and is thus better suited as a quencher for long wavelength dyes like Cy5 compared to other non-anthraquinone quenchers as e.g. dabcyl which on the contrary is more suited for shorter wavelength dyes like e.g. FAM. A structurally related 1,4-diaminoanthraquinone dark quencher called IOWA Black-3.1™ (“IOWA Black”) is available from integrated DNA Technologies (www.idtdna.com). IOWA Black contains an α-aminoaryl group compared to an α-aminoalkyl group in LQ1 thus having a higher extinction cooefficient at higher wavelength compared to LQ1. The LQ1 and IOWA Black phosphoramidite quenchers can only be incorporated at the 5′-end of oligonucleotides when using standard phosphoramidite chemistry on DNA synthesizers and thus cannot be incorporated in the preferred 3′-end of or internally in oligonucleotide molecules. May et al. (Chem. Commun., 2003, 970-971) has prepared the 1,4-diaminoanthraquinone dark quencher molecule LQ2 which was attached to controlled pore glass (CPG) via an amide containing linker and a deoxyribose sugar. With this approach the 1,4-diaminoanthraquinone dark quencher molecule LQ2 can be incorporated into the 3′-end but not internally nor in the 5′-end of the oligonucleotide molecule. When LQ2 modified oligonucleotides are deprotected after oligonucleotide synthesis using standard deprotection conditions (concentrated aqueous ammonia) the LQ2 molecule is cleaved from the oligonucleotide due to the labile amide bonds in the linker. Alternatively, LQ2 modified oligonucleotides can be deprotected using water:methanol:tert-butylamine (2:1:1) or ammonium hydroxide:concentrated aqueous methylamine (1:1) mixtures where the use of the latter mixture results in the cleanest product. However, these procedures require either the use of N-DMF protected G phosphoramidites or N-acetyl protected C phosphoramidites, respectively, e.g. nucleoside phosphoramidites containing non-standard protecting groups.
In summary, there is a need for α-amino substituted anthraquinone dark quenchers that can be easily synthesized, are inexpensive and are capable of being incorporated internally as well as in the 3′-end and 5′-end of oligonucleotide molecules.
Furthermore, a series of quenchers having similar physical properties for e.g. increased static quenching through ground-state complex formation, but distinct spectral properties would be particular advantageous.
WO 2004/026804 A1 was filed before, but was published after, the priority date of the present application. WO 2004/026804 A1 discloses various anthraquinone quencher dyes, their preparation and use.